The primary aim of this proposed research is to further our understanding of the catalytic mechanisms of energy-coupled ATP synthesis during oxidative and photophosphorylation. Because productive substrate binding and product dissociation, not phosphoryl transfer itself, appear to be the energy-dependent steps of ATP synthesis, it is important to understand how these steps are regulated. The ATP synthetase complex of spinach chloroplasts will be investigated with regard to the enzyme's demonstrated ability to change its affinity for product ATP. The mechanism and regulation of the latent ATPase activity of the same enzyme complex also will be studied to determine its relationship to the synthetase activity. Investigations will be done of the reaction mechanism of bacterial membrane ATPase/ATP synthetase, the structure and function of which are similar to that of mitochondrial and chloroplast coupling factors. The main approach will involve oxygen exchange studies (Pi:H2O, ATP:H2O, NTP:H2O). Importantly, analysis for 180 will be done by conversion of phosphoryl groups to trimethyl phosphate followed by mass spectroscopy, analyzing the parent molecular ion for all possible 160/180 species. Keeping all P-O bonds intact has marked advantages over the traditional method of converting Pi to CO2 with regard to the quantity of information obtained. The technique also will be used for measuring reversible ATP cleavage via bound ADP by beta gamma bridge;beta nonbridge positional oxygen exchange in beta gamma bridge-180-labeled ATP. In the long run, the proposed research should contribute to our elucidation of how light and electron transport energy are transformed into chemical energy by the "coupling factor" enzymes, a critical question in overall cellular metabolism.